Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended. IMPORTANCE: Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping.

Avibacterium paragallinarum (A. paragallinarum) is the aetiological agent of infectious coryza (IC) in chickens and characterized by acute respiratory distress and severe drop in egg production. Vaccination is important in the control of IC outbreaks and the efficacy of vaccination is dependent on A. paragallinarum serovars included in the vaccine. Classical serotyping of A. paragallinarum is laborious and hampered by poor availability of antigens and antisera. The haemagglutinin, important in classical serotyping, is encoded by the HMTp210 gene. HMTp210 gene analysis has been shown to have potential as alternative to classical serotyping. The aim of the present study was to further investigate the potential of sequence analyses of partial region 1 of the HMTp210 gene, the HMTp210 hypervariable region and the concatenated sequences of both fragments. For this analysis, 123 HMTp210 gene sequences (field isolates, A. paragallinarum serovar reference strains and vaccine strains) were included. Evaluation of serovar references and vaccine strains revealed a need for critical evaluation, especially within Page serovar B and C. Phylogenetic analysis of HMTp210 region 1 resulted in a separation of Page serovar A, B and C strains. Analysis of the HMTp210 HVR alone was not sufficient to discriminate all nine different Kume serovar references. The concatenated sequences of HMTp210 region 1 and HMTp210 HVR resulted in 14 clusters with a high correlation with Page serovar and with the nine currently known Kume serovars and is therefore proposed as a novel genotyping method that could be used as an alternative for classical serotyping of A. paragallinarum.

Identification, HPG2 Sequence Analysis, and Antimicrobial Susceptibility of Avibacterium paragallinarum Isolates Obtained from Outbreaks of Infectious Coryza in Commercial Layers in Sonora State, Mexico.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Nota de investigación­Análisis de secuencias de la región HPG2 y susceptibilidad antimicrobiana de aislamientos de Avibacterium paragallinarum obtenidos de brotes de coriza infecciosa en aves de postura comerciales en el estado de Sonora, México. Este es el primer informe extenso sobre la identificación y caracterización de aislamientos de Avibacterium paragallinarum (AVP) obtenidos de brotes de coriza infecciosa (IC) de parvadas de ponedoras vacunadas con coriza infecciosa en el estado de Sonora en México. Los aislamientos obtenidos de los brotes de coriza infecciosa durante los años 2007, 2014, 2015, 2017 y 2019 se identificaron mediante una prueba de PCR convencional y el análisis del gene de ARNr 16S, se serotipificaron mediante el método de Page y se genotipificaron mediante el análisis parcial de secuencias descrito recientemente de la región HPG2. Además, se determinaron los perfiles de susceptibilidad a los antimicrobianos mediante la prueba de concentración mínima inhibitoria (MIC) que ha sido mejorada recientemente. La prueba de PCR convencional y los análisis de secuencias del gene ARNr 16S confirmaron que los aislados eran A. paragallinarum. Los resultados de la serotipificación mostraron la participación de aislamientos pertenecientes a los serotipos A, B y C en los brotes de coriza infecciosa. La genotipificación de la región HPG2 reveló la presencia de secuencias del tipo (ST) 1, ST4 y ST11, de los cuales este último también ha sido identificada en Europa. La prueba de susceptibilidad por concentración mínima inhibitoria mostró que todos los aislados analizados eran susceptibles a la mayoría de los antimicrobianos analizados, incluida la eritromicina y la tetraciclina, que son antibióticos importantes para el tratamiento contra la coriza infecciosa. La situación de coriza infecciosa en el estado de Sonora, México, es compleja por la presencia de los serotipos A, B y C. Este hallazgo enfatiza la importancia de la bioseguridad en combinación con la aplicación de los programas de vacunación óptimos en el control de la coriza infecciosa en el estado de Sonora, México.

Identification and characterization of Dutch Avibacterium paragallinarum isolates and the implications for diagnostics.

This study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent. All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

Prevalence of extended-spectrum and AmpC ß-lactamase-producing Escherichia coli in Dutch dairy herds.

This study was conducted to assess: (1) a change in between-herd prevalence of extended-spectrum and AmpC ß-lactamase-producing Escherichia coli (ESBL/AmpC-EC) between 2011 and 2013, the period during which the antimicrobial policy in animal husbandry in the Netherlands changed significantly, and (2) the prevalence of ESBL/AmpC-EC in individual calves, young stock, and dairy cows in the Netherlands. In 196 randomly selected conventional dairy herds, faecal samples were collected from calves (maximum n = 15), and randomly selected young stock (n = 5) and dairy cows (n = 15). Additionally, fresh faecal samples were collected from five different places on the floors where the dairy cows were housed. Samples were screened for E. coli with non-wild type susceptibility for cefotaxime and isolates were phenotypically confirmed as ESBL/AmpC-producing by disc diffusion, using cefotaxime and ceftazidime with and without clavulanic acid, and cefoxitin. Samples containing ESBL/AmpC-EC were examined semi-quantitatively. In 59.6% of the dairy herds one or more samples tested positive for ESBL/AmpC-EC. The between-herd prevalence based on floor samples in 2013 (18.0%) was significantly lower than the prevalence in 2011 based on comparable samples (32.7%). The individual animal prevalence of ESBL/AmpC-EC, with a minimum shedding level of 103 cfu/g of faeces, was 19.3% in calves, 0.9% in young stock, and 0.8% in dairy cows. Although ESBL/AmpC-EC was found in the majority of dairy herds, the herd prevalence declined significantly between 2011 and 2013. Calves were found to have both, a much higher individual animal prevalence and a higher level of shedding than young stock and cows.

Outbreaks of canine distemper in Dutch and Belgian mink farms.

BACKGROUND: Vaccination of farmed minks against canine distemper virus (CDV) has proved to be very effective. In the Netherlands, vaccination of farmed minks against CDV was mandatory until the closure of the local agricultural product boards at the end of 2014. OBJECTIVES: To describe the first documented outbreaks of CD in Dutch mink farms since the closure of the agricultural product boards, as well as an outbreak in Belgium, with special attention to genotyping of the isolates. METHODS: A full post-mortem was performed on three carcasses per submission from farms A-C and on two carcasses from farm D. Molecular detection with subsequent typing was performed on eleven samples originating from four different farms. To assess genetic diversity partial sequences of the H gene of CDV were compared based on phylogenetic analysis. RESULTS: In 2017, there was a sudden series of CD outbreaks affecting four mink farms in the Netherlands (A-C) and Belgium (D). Gross, histologic and immunohistochemical findings were similar. There was a degree of genetic similarity between the viruses on farms A and D (98.5%) and between the viruses on farms B and C (97.3%), but the viruses from farms A and D belonged to a different clade than the viruses from farms B and C. Higher mortalities were reported in white and pastel minks. CONCLUSIONS: Findings indicated that the difference in severity of the outbreaks was partially related to the genetic composition of the farm populations. Vaccination against CDV on Dutch and Belgian mink farms seems warranted.

Postvaccination wounds associated predominantly with Arcanobacterium phocae in mink (Neovison vison) at three mink farms.

BACKGROUND: The emerging skin disease fur animal epidemic necrotic pyoderma (FENP) has been attributed to infection with Arcanobacterium phocae (ABP). The exact pathogenesis and risk factors of FENP have yet to be elucidated. ANIMALS: Three mink from each of three different mink farms (A-C) with postvaccination skin wounds at the vaccination site and six mink from an unaffected mink farm (D) that had used the same vaccine batch and vaccination site (hind leg). METHODS AND RESULTS: All mink from farms A-C had severe necrotizing to necropurulent dermatitis where they were vaccinated intramuscularly in the hind leg. ABP was the sole bacterium cultured from six of nine wounds. Using 16S-23S rDNA intergenic spacer region and BOX-PCR, the ABP isolates from these wounds were indistinguishable from isolates originating from several cases of FENP. CONCLUSIONS AND CLINICAL IMPORTANCE: This is the first report of FENP-like lesions at the site of vaccination, in the days following the procedure, associated with ABP. At farms with FENP vaccination, procedures should be considered carefully.